Ceramide analogs, process for their preparation and their use as antitumor agents

ABSTRACT

The present invention is directed to ceramide analog compounds of general formula (I) the process for their preparation and use for the preparation of pharmaceutical formulations for the treatment of tumors

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/031,692 filed Jan. 22, 2002 now U.S. Pat. No. 6,956,109, which is a35 U.S.C. § 371 U.S. national stage application of PCT ApplicationPCT/EP00/07023, filed Jul. 21, 2000, which claims priority to and thebenefit of Italian Patent Application No. F199A000169, filed Jul. 22,1999, all of which are hereby incorporated by reference in theirentirety.

FIELD OF THE INVENTION

The present invention concerns the ceramide analog compounds of thegeneral formula (I) specified below, their corresponding preparationprocess, and their use in the preparation of pharmaceutical formulationswith an antitumor effect.

STATE OF THE ART

Ceramides are lipids composed of a fatty acid and sphingosine joinedtogether by an amide link; they are generated by sphingomyelin, asphingolipid occurring in the membranes of eukaryote cells due to theaction of the enzyme sphingomyelinase, or they are synthesized by theaction of the enzyme ceramide synthetase.

Sphingolipids such as sphingomyelin have always been considered asstable and metabolically inactive structural components of themembranes. It is only in the last decade that it has been demonstrated,instead, that sphingolipids have an active role in the mechanismsregulating cell response to exogenous stimuli, as well as in regulatingcell growth, differentiation, transformation and adhesion.

It has also recently been demonstrated that the products of thedemolition of sphingolipids, i.e. ceramides and sphingosine, play animportant part in regulating the transmission mechanisms of the signalscontrolled by the membrane sphingolipids (Teruyuki Sakai et al., ExpOpin Ther Patents [1988] 8 [12]: 1673–1682). In particular, thedistinctive characteristic of these products seems to be theirinvolvement in the antiproliferative mechanisms of cell regulation, suchas cell growth inhibition, the induction of cell differentiation andprogrammed cell death, or apoptosis.

Apoptosis has recently been the object of numerous studies (e.g. Ross A.Kinloch et al., TiPS, January 1999 [20]: 35–42), because this phenomenonlends itself to pharmacological “manipulation”: in fact, a reduction inthe frequency of the onset of cell apoptosis can have severepathological consequences and facilitate tumor growth, hence thetherapeutic potential of all those compounds that are capable ofstimulating apoptosis.

From in-depth studies it has emerged that the ceramides in the cellmembranes act as intracellular “effectors” of apoptosis, and thereforeas potential inhibitors of tumor growth.

In order to boost this capacity of the endogenous ceramidespharmacologically, the ideal strategy seems to be to develop endogenousceramide analogs that mimic their effects, are stable in relation tometabolization of the sphingosine ceramide and have an inhibitory effecton the ceramidase in order to prevent the generation of sphingosine,which represents a factor that stimulates proliferation, starting fromthe endogenous sources of ceramides.

Such ceramide analogs should also have the capacity to penetrate thecell membrane.

There is consequent a need for ceramide analog compounds that arecapable of crossing the cell membranes, penetrating inside the cells andmimicking the various properties of the ceramides, and particularly thatof inducing apoptosis in human cancer cells.

SUMMARY OF THE INVENTION

The Applicant has now surprisingly discovered that the ceramide analogcompounds of formula (I):

wherein:

-   X₁ and X₂ are selected between O and S;-   R₁ and R₂ are selected between —(CH₂)₁₃CH₃ and alkyl or alkylene    groups with from 2 to 6 carbon atoms, linear or branching,    unsubstituted or substituted with one or more substituents selected    among aromatic, primary, secondary and tertiary aminic, quaternary    ammonium, carboxylic, hydroxylic, polyoxyalkylic and ethereal    groups, aminoacids, halogen atoms or saccharidic portions, providing    that between R₁ and R₂ only one is always —(CH₂)₁₃CH₃,-   R₃ and R₄ are selected between H and alkyl or alkylene groups with    from 2 to 6 carbon atoms, linear or branching, unsubstituted or    substituted with one or more substituents selected among aromatic,    primary, secondary and tertiary aminic, quaternary ammonium,    carboxylic, hydroxylic, polyoxyalkylic and ethereal groups,    aminoacids, halogen atoms or saccharidic portions,-   are capable of penetrating inside the biological membranes and    effectively inducing apoptosis of the cancer cells.

The compounds of the general formula (I) considered in the presentinvention have therefore proved suitable for the preparation ofpharmaceutical formulations for the treatment of tumors.

The object of the present invention is therefore represented by thecompounds of the general formula (I), their corresponding preparationprocess, and their use in the preparation of pharmaceutical formulationsfor use in the treatment of tumors. The characteristics and advantagesof the compounds of the general formula (I) according to the presentinvention will be illustrated in detail in the following description.

DETAILED DESCRIPTION OF THE INVENTION

The present invention refers to the compounds of the general formula(I), as defined above. Said compounds (I) have proved capable ofpenetrating inside the biological membranes and effectively inducing theapoptosis of cancer cells. The following compounds have provedparticularly effective and highly cytotoxic:

-   -   compound of formula (I) where X₁=S, X₂=O, R₁=—(CH₂)₁₃CH₃,        R₂=ethyl, and R₃=R₄=H [compound (3)];    -   compound of formula (I) where X₁=X₂=O, R₁=—(CH₂)₁₃CH₃, R₂=ethyl,        and R₃=R₄=H [compound (4)];    -   compound of formula (I) where X₁=X₂=O, R₁ =—(CH₂)₁₃CH₃,        R₂=n-propyl, and R₃=R₄=H [compound (6)];    -   compound of formula (I) where X₁=X₂=O, R₁=—(CH₂)₁₃CH₃,        R₂=i-butyl, and R₃=R₄=H [compound (10)];

The present compounds of formula (I) can be conveniently prepared byprocesses well known in the art. For example, a process for thepreparation of the present compounds of formula (I) wherein R₃=R₄=Hincludes the following steps:

-   i) reaction of the ethyl ester (II) with acid chloride (III) to    obtain the β-ketoester of formula (IV):

-   ii) reaction of the β-ketoester of formula (IV) with thiourea (V) to    obtain the compound of formula (I) where X₁=S, X₂=O:

-   iii) reaction compound of formula (I) where X₁=S, X₂=O, with    refluxed chloroacetic acid to obtain the compound of formula (I)    wherein X₁=X₂=O:

wherein X₁, X₂, R₁ and R₂ have above-specified meanings.

Step i) of the said process is generally carried out in an organicsolvent, such as THF, at a temperature of 0° C. Said reaction ispreferably carried out in an inert gas atmosphere.

The reaction product of formula (IV) can be recovered from the reactionmixture by addition of a saturated NH₄Cl solution and subsequentextraction with diethyl ether.

Step ii) of the present process is carried out by means of the additionof thiourea in ethanol and sodium ethoxide on the raw reaction productcoming from step i), without the need for any purification. In step ii)temperature is preferably maintained around 90° C. The reaction productis generally recovered from the reaction mixture by acidification at pH2, e.g. by adding conc. HCl, and filtration of the resultingprecipitate, which can be purified, if necessary, by washing withacetone.

The reaction product obtained in step ii) can be further purified bychromatography on silica gel, preferably using a mixture of ethylacetate and petroleum ether in proportions of 2:1 as an eluant

Step iii) of the process according to the above procedure is generallycarried out by adding chloroacetic acid to the product coming from stepii), e.g. in the form of a 10% aqueous solution, and reflux heated. Thecrude residue thus obtained can then be purified by washing withabsolute ethanol and then with diethyl ether.

The product coming from step iii) can be further purified bychromatography on silica gel, preferably using a mixture of ethylacetate and hexane in proportions of 1:2 as an eluant.

The present compounds of formula (I) wherein R₃ and/or R₄ are differentfrom H, can be prepared from the β-ketoester of formula (IV) or from thecompounds of formula (I) wherein R₃=R₄=H, obtained for example asexplained above, by means of well-known processes.

Other processes for the preparation of the present formula (I) compoundsare disclosed in the examples.

The compounds of formula (I) according to the present invention can beformulated with pharmaceutically acceptable excipients and/or diluentsin order to prepare pharmaceutical formulations suitable for thetreatment of tumor pathologies.

The following examples are given as a partial illustration of thepresent invention.

EXAMPLE 1

Preparation of the Compound of Formula (I) Where X₁=S, X₂=O,R₂=—(CH₂)₁₃CH₃, R₁=ethyl, and R₃=R₄=H [Compound (1)]

A solution prepared by dissolving 0.37 g of ethyl butyrate in 2 ml ofanhydrous tetrahydrofuran (THF) is added drop by drop, at a temperatureof 0° C. and in an argon gas atmosphere, to 1.9 ml of a 2M solution oflithiodiisopropylamine (LDA) in anhydrous THF. After 30 minutes ofagitation at 0° C., the reaction mixture is added to a solution obtainedby dissolving 1 g of pentadecanol chloride (3.8 mmol) in 5 ml ofanhydrous THF, previously cooled to 0° C. The resulting mixture isconstantly agitated at room temperature for 12 hours, then added to asaturated solution of NH₄Cl. The organic phase is separated from theaqueous phase, then extracted with diethyl ether. The organic extractsare combined, washed with a saturated aqueous solution of NaCl, driedwith anhydrous Na₂SO₄ and then evaporated until dry to provide a cruderesidue (1.20 g) composed almost exclusively of β-ketoester (IV) whereR₂=—(CH₂)₁₃CH₃ and R₁=ethyl [¹H-NMR (CDCl₃, 200 MHz) δ 0.83–0.94 (m,6H), 1.07 (t, 3H, J=7.4 Hz), 1.15–1.36 (m, 24H), 1.81–2.02 (m, 2H),2.11–2.57 (m, 2H), 3.34 (t, 1H, J=7.3 Hz), 4.15 (q, 2H, J=7.3 Hz). MSm/e 340 M⁺].

The resulting crude residue (1.20 g) containing the β-ketoester (IV)where R₂=—(CH₂)₁₃CH₃ and R₁=ethyl, is dissolved in 20 ml of absoluteethanol and then added to 3.61 g of thiourea (47.5 mmol) and 6.47 g ofsodium ethoxide (95.1 mmol). The mixture is agitated for 60 minutes at90° C. After cooling to room temperature, the reaction mixture isfiltered and the filtrate is evaporated until dry; the residue thusobtained is then restored with a mixture of water and THF in proportionsof 10:1 until it has become completely soluble. The solution, cooled to0° C., is acidified to pH 2 with conc. HCl; the precipitate thatdevelops is filtered and washed with small quantities of acetone andprovides a crude residue that is purified by chromatography on silicagel using ethyl acetate and petroleum ether in proportions of 2:1 as aneluant, finally obtaining 290 mg (0.82 mmol; yield =26%) of the requiredcompound of formula (I) (m.p. =167–169° C.; [¹H-NMR (CDCl₃, 200 MHz) δ0.89 (t, 3H, J=6.2 Hz), 1.09 (t, 3H, J=7.4 Hz), 1.17–1.36 (m, 24H),2.34–2.49 (m, 4H), 8.88 (br, 1H, D₂O exchangeable), 9.81 (br, 1H, D₂Oexchangeable); MS m/e 352 M⁺).

EXAMPLE 2

Preparation of the Compound of Formula (I) Where X₁=X₂=O,R₂=—(CH₂)₁₃CH₃, R₁=ethyl, and R₃=R₄=H [Compound (2)]

160 mg (0.45 mmol) of the product (1) obtained as described in Example 1are added to 11.4 ml of a 10% aqueous solution of chloroacetic acid andthe mixture thus obtained is reflux heated for 12 hours. The resultingprecipitate is then filtered, washed first with absolute ethanol, thenwith diethyl ether, to obtain a crude residue that, after purificationby chromatography on silica gel using a mixture of ethyl acetate andhexane in proportions of 1:2 as an eluant, gave rise to 48 mg (0.14mmol, yield =32%) of the required pure compound (m.p. =132–134° C.;[¹H-NMR (CDCl₃, 200 MHz) δ 0.87 (t, 3H, J=6.2 Hz), 1.06 (t, 3H, J=7.4Hz), 1.15–1.36 (m, 24H), 2.31–2.49 (m, 4H), 9.06 (br, 1H, D₂Oexchangeable), 9.89 (br, 1H, D₂O exchangeable); MS m/e 336 M⁺).

EXAMPLE 3

Preparation of the Compound of Formula (I) Wherein X₁=S, X₂=O,R₁=—(CH₂)₁₃CH₃, R₂=ethyl, and R₃=R₄=H [Compound (3)]

A solution obtained by dissolving 1 g of ethyl palmitate (3.52 mmol) in3 ml of anhydrous THF is added drop by drop, at a temperature of 0° C.in an argon gas atmosphere, to 2.1 ml of a 2M solution oflithiodiisopropylamine (LDA) in anhydrous THF. After 30 minutes ofagitation at 0° C., the reaction mixture is added to a solution obtainedby dissolving 2.39 g (4.23 mmol) of propionyl chloride in 5 ml ofanhydrous THF. The resulting mixture is constantly agitated at roomtemperature for 12 hours, then added to a saturated solution of NH₄Cl.The organic phase is separated from the aqueous phase, then extractedwith diethyl ether. The organic extracts are combined, washed with asaturated aqueous solution of NaCl, dried with anhydrous Na₂SO₄ and thenevaporated until dry to provide a crude residue (1.31 g) composed almostexclusively of β-ketoester (IV) where R₁=—(CH₂)₁₃CH₃ and R₂=ethyl.[¹H-NMR (CDCl₃, 200 MHz) δ 0.79–0.92 (m, 6H), 1.11 (t, 3H, J=7.6 Hz),1.17–1.39 (m, 24H), 148–1.62 (m, 2H), 226 (q, 2H, J=7.6 Hz), 3.36 (t,1H, J=7.3 Hz), 4.15 (q, 2H, J=7.2 Hz); MS m/e 340 M⁺]. 1.31 g of theresulting crude residue containing the β-ketoester (IV) whereR₁=—(CH₂)₁₃CH₃ and R₂=ethyl, is dissolved in 20 ml of absolute ethanoland then added to 4.01 g of thiourea (52.8 mmol) and 7.18 g of sodiumethoxide (105.6 mmol). The mixture is agitated for 60 minutes at 90° C.After cooling to room temperature, the reaction mixture is filtered andthe filtrate is evaporated until dry; the residue thus obtained is thentreated with a mixture of water and THF in proportions of 10:1 until ithas become completely soluble. The solution is cooled to 0° C. andacidified to pH 2 with conc. HCl; the precipitate that develops due toacidification is filtered and washed with small quantifies of acetoneand provides a crude residue that is purified by chromatography onsilica gel using ethyl acetate and petroleum ether in proportions of 2:1as an eluant, finally obtaining 310 mg (0.88 mmol; yield =25%) of aproduct that coincides with the required pure compound 3 (m.p. =100–102°C.; [H-NMR (CDCl₃, 200 MHz) δ 0.88 (t, 3H, J=6.4 Hz), 1.01 (t, 3H, J=7.4Hz), 1.18–1.38 (m, 24H), 2.35 (t, 2H, J=7.4 Hz), 2.48 (q, 2H, J=7.6 Hz),9.08 (br, 1H, D₂O exchangeable), 9.73 (br, 1H, D₂O exchangeable); MS m/e352 M⁺).

EXAMPLE 4

Preparation of the Compound of Formula (I) Wherein X₁=X₂=O, R₁=—(CH₂)₁₃CH₃, R₂=ethyl, and R₃=R₄=H [Compound (4)]

160 mg (0.45 mmol) of the compound (3) obtained as described in Example3 are added to 11.4 ml of a 10% aqueous solution of chloroacetic acidand the mixture thus obtained is reflux heated for 12 hours. Theresulting precipitate is then filtered, washed first with absoluteethanol, then with diethyl ether, to obtain a crude residue that, afterpurification by chromatography on silica gel using a mixture of ethylacetate and hexane in proportions of 1:2 as an eluant, gave rise to 57mg (0.17 mmol, yield =38%) of the compound (4) (m.p. =110–112° C.;[¹H-NMR (CDCl₃, 200 MHz) δ 0.89 (t, 3H, J=6.4 Hz), 1.02 (t, 3H, J=7.4Hz), 1.12–1.42 (m, 24H), 2.34 (t, 2H, J=7.2 Hz), 2.49 (q, 2H, J=7.6 Hz),9.15 (br, 1H, D₂O exchangeable), 9.53 (br, 1H, D₂O exchangeable); MS m/e336 M⁺).

EXAMPLE 5

Preparation of the Compound of Formula (I) Wherein X₁=S, X₂=O,R₁=—(CH₂)₁₃CH₃, R₂=n-propyl, and R₃=R₄=H [Compound (5)]

Compound (5) was prepared following a procedure similar to the onedescribed in Example 3, obtaining a product which resulted in: MS m/e366 M⁺.

EXAMPLE 6

Preparation of the Compound of Formula (I) Wherein X₁=X₂=O,R₁=—(CH₂)₁₃CH₃, R₂=n-propyl, and R₃=R₄=H [Compound (6)]

Compound (6) was prepared following a procedure similar to the onedescribed in Example 4, obtaining a product which resulted in: MS m/e350 M⁺.

EXAMPLE 7

Preparation of the Compound of Formula (I) Wherein X₁=S, X₂=O,R₂=—(CH₂)₁₃CH₃, R₂=n-butyl, and R₃=₄=H [Compound (7)]

Compound (7) was prepared following a procedure similar to the onedescribed in Example 3, obtaining a product which resulted in: MS m/e380 M⁺.

EXAMPLE 8

Preparation of the Compound of Formula (I) Wherein X₁=X₂=O,R₁=—(CH₂)₁₃CH₃, R₂=n-butyl, and R₃=R₄=H [Compound (8)]

Compound (8) was prepared following a procedure similar to the onedescribed in Example 4, obtaining a product which resulted in: MS m/e364 M⁺.

EXAMPLE 9

Preparation of the Compound of Formula (I) Wherein X₁=S, X₂=O,R₁=—(CH₂)₁₃CH₃, R₂=i-butyl, and R₃=R₄=H [Compound (9)]

Compound (9) was prepared following a procedure similar to the onedescribed in Example 3, obtaining a product which resulted in: MS m/e380 M⁺.

EXAMPLE 10

Preparation of the Compound of Formula (I) Wherein X₁=X₂=O,R₁=—(CH₂)₁₃CH₃, R₂=i-butyl, and R₃=R₄=H [Compound (10)]

Compound (10) was prepared following a procedure similar to the onedescribed in Example 4, obtaining a product which resulted in: MS m/e364 M⁺.

EXAMPLE 11

Preparation of the Compound of Formula (I) Wherein X₁=S, X₂=O,R₁=—(CH₂)₁₃CH₃, R₂=neopentyl, and R₃=R₄=H [Compound (11)]

Compound (11) was prepared following a procedure similar to the onedescribed in Example 3, obtaining a product which resulted in: MS m/e394 M⁺.

EXAMPLE 12

Preparation of the Compound of Formula (I) Wherein X₁=X₂=O,R₁=—(CH₂)₁₃CH₃, R₂=neopentyl, and R₃=R₄=H [Compound (12)]

Compound (12) was prepared following a procedure similar to the onedescribed in Example 4, obtaining a product which resulted in: MS m/e378 M⁺.

EXAMPLE 13

Preparation of the Compound of Formula (I) Wherein X₁=S, X₂=O,R₁=—(CH₂)₁₃CH₃, R₂=2-phenyl-ethyl, and R₃=R₄=H [Compound (13)]

Compound (13) was prepared following a procedure similar to the onedescribed in Example 3, obtaining a product which resulted in: MS m/e428 M⁺.

EXAMPLE 14

Preparation of the Compound of Formula (I) Wherein X₁=X₂=O,R₁=—(CH₂)₁₃CH₃, R₂=2-phenyl-ethyl, and R₃=R₄=H [Compound (14)]

Compound (14) was prepared following a procedure similar to the onedescribed in Example 4, obtaining a product which resulted in: MS m/e412 M⁺.

EXAMPLE 15

Preparation of the Compound of Formula (I) Wherein X₁=S, X₂=O,R₁=—(CH₂)₁₃CH₃, R₂=—(CH₂)₃NH₂ and R₃=R₄=H [Compound (15)]

Compound (15) was prepared following the procedure described in theabove Scheme 1.

Synthesis of β-ketoester (32). 2.4 g (10 mmol) of 4-phthalimidoburyricacid (29) (prepared as described in G. Talbot, R. Gaudry, L. BerlinguetCan. J. Chem. 1958, 36, 593–596) was dissolved in 7.5 ml of SOCl₂ andthe mixture was refluxed under nitrogen for 3 hours. Excess of SOCl₂ wasthen removed under a nitrogen flow and the resulting acid chloride (30)was used in the next step without further purification. Separately, asolution of ethyl palmitate (31) (1.47 g, 5.16 mmol) in anhydrous THF(6.5 ml) was slowly added to a 1.0 M solution of lithiumbis(trimethylsilyl)amide (LHMDS) in THF (6.2 ml, 6.2 mmol) cooled at−20° C. and the resulting mixture was stirred for additional 20 minutes.Acid chloride (30) previously prepared as described above, was dissolvedin anhydrous THF (10 ml), cooled at −20° C., and added via cannula tothe solution containing (31) and LHMDS at the same temperature. Themixture was stirred at −20° C. for 30 minutes and then at roomtemperature for 2 hours. The reaction was quenched with a saturatedaqueous solution of ammonium chloride and extracted with ethyl acetate.The organic layer was dried over sodium sulfate and concentrated undervacuum. The crude residue was purified by silica gel columnchromatography using hexane-ethyl acetate (8:2) as the eluant, to obtain0.95 g (1.9 mmol, 37% yield) of pure β-ketoester (32) as a colorlessoil: ¹H NMR (CDCl₃, 200 MHz) δ 0.87 (t, 3H, J=6.4 Hz), 1.23 (t, 3H,J=7.3 Hz), 1.24 (bs, 24H), 2.05 (quintet, 2H, J=7.2 Hz), 2.24–2.34 (m,4H), 2.51 (t, 2H, J=7.6 Hz), 3.78 (t, 1H, J=6.9 Hz), 4.12 (q, 2H, J=7.1Hz), 7.69–7.73 (m, 2H), 7.82–7.87 (m, 2H); MS (FAB⁺) m/z 500 (M+H)⁺.

Synthesis of thiouracil (15). β-Ketoester (32) (0.12 g, 0.24 mmol) wasdissolved in 2 ml of absolute ethanol. Thiourea (0.024 g, 0.33 mmol) andpotassium t-butoxyde (0.028 g, 0.25 mmol) were added and the resultingmixture was refluxed for 5 hours. The mixture was then cooled to roomtemperature and the solvent was removed under vacuum. The residue wastreated with 20 ml of water and neutralized with an aqueous solution ofacetic acid 0.5 N. The product was extracted with ethyl acetate and theorganic layer was washed with brine, dried over anhydrous sodium sulfateand concentrated under vacuum. The crude residue was then redissolved in3 ml of ethanol, treated with 0.06 ml of hydrazine monohydrate (1.3mmol), and the mixture was refluxed overnight. The resulting suspensionwas cooled to room temperature. The white solid was collected byfiltration, washed with small portions of ethyl acetate, and dried undervacuum, to give 51 mg (0.13 mmol, 54% yield) of product (15): m.p.123–125° C.; ¹H NMR (CDCl₃, 200 MHz) δ 0.88 (t, 3H, J=6.4 Hz), 1.26 (bs,24H), 1.77 (m, 2H), 2.29–2.45 (m, 6H), 8.87 (bs, 1H), 9.19 (bs, 1H); MS(FAB⁺) m/z 381 (M+H)⁺.

EXAMPLE 16

Preparation of the Compound of Formula (I) where X₁=S, X₂=O,R₁=—(CH₂)₁₃CH₃, R₂=—(CH₂)₃OSiPh₂t-Bu and R₃=R₄=H [Compound (16)]

Compound (16) was prepared following the procedure described in thefollowing

Synthesis of methyl ester (34). A solution of acid (33) (1.15 g, 3.36mmol) (prepared as in: A. G. M. Barrett, J. A. Flygare J. Org. Chem.1991, 56, 638–642) in methanol (25 ml) was treated with 1.62 g (8.44mmol) of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride(EDC). The resulting solution was stirred under nitrogen at roomtemperature for 3.5 hours. The solvent is then removed under vacuum andthe residue was diluted with chloroform (100 ml) and water (50 ml). Theorganic layer was washed with brine, dried over sodium sulfate andconcentrated under vacuum. The crude residue was purified by silica gelcolumn chromatography using hexane-ethyl acetate (9:1) as the eluant, toobtain 0.59 g (1.6 mmol, 49% yield) of pure ester (34) as a colorlessoil: ¹H NMR (CDCl₃, 200 MHz) δ 1.05 (s, 9H), 1.88 (tt, 2H, J=7.7, 5.9Hz), 2.47 (t, 2H, J=7.5 Hz), 3.66 (s, 3H), 3.68 (t, 2H, J=6.0 Hz),7.37–7.42 (m, 6H), 7.63–7.68 (m, 4H).

Synthesis of β-ketoester (36). A solution of t-butyl acetate (35) (4.24g, 36.5 mmol) in anhydrous THF (40 ml) previously cooled at −78° C. wasadded drop by drop via is cannula under argon to a 1M solution of LHMDSin THF (51.5 ml, 51.5 mmol). To the resulting solution, previouslystirred at the same temperature for 30 minutes, was added drop by dropvia cannula another solution of methyl ester (34) (4.07 g, 11.4 mmol) inanhydrous THF (20 ml) at −78° C. The reaction mixture was stirred underargon for 20 minutes at the same temperature, and then 3 more hours atroom temperature. The reaction was quenched with 400 ml of saturatedaqueous solution of ammonium chloride and extracted with diethyl ether(2×300 ml). The organic layer was dried over anhydrous sodium sulfateand concentrated under vacuum. The crude residue was purified by silicagel column chromatography using hexane-diethyl ether (8:2) as theeluant, to obtain 2.8 g (6.4 mmol, 56% yield) of pure β-ketoester (36)as a colorless oil: ¹H NMR (CDCl₃, 200 MHz) δ 1.04 (s, 9H), 1.46 (s,9H), 1.84 (quintet, 2H, J=6.7 Hz), 2.66 (t, 2H, J=7.3 Hz), 3.34 (s, 2H),3.67 (t, 2H, J=6.0 Hz), 7.37–7.43 (m, 6H), 7.62–7.67 (m, 4H): MS (FAB⁺)m/z 441 (M+H)⁺, 385 (M+H-isobutene)⁺.

Synthesis of alkylated β-ketoester (37). A solution of β-ketoester (36)(2.79 g, 6.34 mmol) in anhydrous 1,2-dimethoxyethane (DME) (17 ml) wasadded to a solution of potassium tert-butoxide (0.85 g, 6.97 mmol) inanhydrous DME (7 ml). The resulting solution was stirred at roomtemperature for 20 minutes, after which time 1.7 ml (1.6 g, 5.7 mmol) of1-bromotetradecane were added. The reaction mixture was stirred at 80°C. for 2 hours. The reaction was quenched with 150 ml of a saturatedaqueous solution of ammonium chloride and extracted with diethylether(2×300 ml). The organic layer was washed with brine, dried over sodiumsulfate and concentrated under vacuum. The crude residue was purified bysilica gel column chromatography using hexane-diethyl ether (9:1) as theeluant, to obtain 1.16 g (1.82 mmol, 32% yield) of pure mono-alkylatedproduct (37) as a colorless oil: ¹H NMR (CDCl₃, 200 MHz) δ 0.88 (t, 3H,J=6.2 Hz), 1.04 (s, 9H), 1.25 (bs, 24H), 1.43 (s, 9H), 1.76–1.89 (m,4H), 2.64 (td, 2H, J=7.3, 4.4 Hz), 3.13 (t, 1H, J =7.3 Hz), 3.66 (t, 2H,J=6.0 Hz), 7.34–7.43 (m, 6H), 7.62–7.67 (m, 4H); MS (FAB⁺) m/z 581(M+H-isobutene)⁺, 563 (M−tBuO)⁺.

Synthesis of thiouracil (16). A solution containing alkylatedβ-ketoester (37) (1.16 g, 1.82 mmol) in absolute ethanol (24 ml) in ascrew-cap sealed vial was treated first with 0.19 g (2.6 mmol) ofthiourea and then with 0.25 g (2.0 mmol) of potassium tert-butoxide. Theresulting solution was stirred at 100° C. for 6 hours. The solvent wasthen removed under vacuum. The residue was diluted with water andneutralized to pH=6–7 with 0.5 N acetic acid. The product was extractedwith ethyl acetate. The organic layer was washed with brine, dried overanhydrous sodium sulfate and concentrated under vacuum. The cruderesidue was purified by silica gel column chromatography usinghexane-diethyl ether (8:2) as the eluant, to obtain 0.52 g (0.84 mmol,46% yield) of pure thiouracil product (16) as a colorless oil: ¹H NMR(CDCl₃, 200 MHz) δ 0.87 (t, 3H, J=6.2 Hz), 1.10 (s, 9H), 1.24 (bs, 24H),1.74 (quintet, 2H, J=6.8 Hz), 2.34 (t, 2H, J=7.4 Hz), 2.60 (t, 2H, J=7.5Hz), 3.74 (t, 2H, J=5.8 Hz), 7.40–7.46 (m, 6H), 7.66–7.70 (m, 4H), 9.29(bs, 1H), 9.55 (bs, 1H); MS (FAB⁺) m/z 547 (M+H-NHC(S)NH)⁺.

EXAMPLE 17

Preparation of the Compound of Formula (I) Where X₁=S, X₂=O,R₁=—(CH₂)₁₃CH₃, R₂=—(CH₂)₃OH and R₃=H [Compound (17)]

According to the above Scheme 3 the silyl ether 16 (0.16 g, 0.26 mmol)was treated with 0.8 ml of a 1 M solution of tetrabutylammonium fluoride(TBAF) in THF (0.8 mmol) under argon at room temperature for 2 hours.The solvent was then removed under vacuum and the residue was extractedwith ethyl acetate. The organic layer was washed with brine, dried oversodium sulfate and concentrated under vacuum. The crude residue waspurified by silica gel column chromatography using hexane-ethyl acetate(1:1) as the eluant, to obtain 0.079 g (0.21 mmol, 81% yield) of puredeprotected alcohol (17) as a white solid: m.p. 128–130° C.; ¹H NMR(CDCl₃, 200 MHz) δ 0.88 (t, 3H, J=6.7 Hz), 1.25 (bs, 24H), 1.91(quintet, 2H, J=6.1 Hz), 2.37 (pseudo t, 2H, J=7.4 Hz), 2.67 (pseudo t,2H, J=6.3 Hz), 3.84 (t, 2H, J=5.6 Hz), 9.16 (bs, 1H), 10.52 (bs, 1H); MS(EI, 70 eV) m/z 382 (M)⁺, 365 (M—OH)⁺, 323 (M—NHC=S)⁺.

EXAMPLE 18

Preparation of the Compound of Formula (I) Where X₁=S, X₂=O,R₁=—(CH₂)₁₃CH₃, R₂=—(CH₂)₃OC(O)CH₂NH—Cbz and R₃=R₄=H [Compound (18)]

According to scheme 4 a solution of the alcohol (17) (0.038 g, 0.099mmol) in anhydrous THF (2.5 ml) was sequentially treated with 0.031 g(0.15 mmol) of N-carbobenzyloxyglycine (N-Cbz-Gly), 0.034 g (0.18 mmol)of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC),and 0.0012 g (0.0096 mmol) of 4-(dimethylamino)pyridine (DMAP). Themixture was stirred at room temperature for 5 hours under argon. Thesolvent was removed under vacuum and the residue was purified by silicagel column chromatography using hexane-ethyl acetate (1:1) as theeluant, to obtain 0.052 g (0.091 mmol, 92% yield) of product (18) as athick syrup: ¹H NMR (CDCl₃, 200 MHz) δ 0.87 (t, 3H, J=6.6 Hz), 1.25 (bs,24H), 1.91 (m, 2H). 2.31 (t, 2H, J=7.7 Hz), 2.47 (t, 2H. J=7.7 Hz), 4.07(d, 2H, J=5.9 Hz), 4.27 (t, 2H, J=5.2 Hz), 5.24 (s, 2H), 5.52 (t, 1H,J=5.8 Hz), 7.31–7.38 (m, 5H), 10.09 (bs, 1H), 10.85 (bs 1H); MS (FAB⁺)m/z 574 (M+H)⁺, 532 (M—C(S)+H)⁴.

EXAMPLE 19

Preparation of the Compound of Formula (I) Where X₁=S, X₂=O,R₁=—(CH₂)₁₃CH₃, R₂=—(CH₂)₃OC(O)CH₂NH₂ and R₃=R₄H [Compound (19)

According to the above scheme 5 a solution of Cbz-protected compound(18) (0.032 g, 0.055 mmol) in trifluoroacetic acid (1 ml) was treatedwith 0.22 mmol of freshly prepared boron tris(trifluoroacetate)(prepared as reported in: J. Pless, W. Bauer Angew. Chem. Int. Ed. 1973,12, 147–148) at 0° C. under argon. The mixture was stirred for 1 hour atthe same temperature and overnight at room temperature. The solvent wasremoved under vacuum and the residue was purified by silica gel columnchromatography using a mixture dichloromethane: acetone 7:3 as theeluant, to obtain 0.020 g (0.045 mmol, 82% yield) of product (19) as athick syrup: ¹H NMR (CDCl₃, 200 MHz) δ 0.87 (t, 3H, J=6.4 Hz), 1.25 (bs,24H), 1.72 (m, 2H), 2.32 (m, 2H), 2.63 (m, 2H), 3.61 (t, 2H, J=7.0 Hz),4.30 (t, 2H, J=6.6 Hz); Ms (FAB⁺) m/z 365 (M-NHC(S)NH)⁺.

EXAMPLE 20

Preparation of the Compound of Formula (I) Where X₁=S, X₂=O,R₁=—(CH₂)₁₃CH₃,

and R₃ R₄H [Compound (20)]

According to the above scheme 6 a solution of alcohol (17) (0.120 g,0.314 mmol) in anhydrous THF (10 ml) was treated sequentially withN-(9-Fluorenylmethoxycarbonyl)-L-aspartic acid tert-butyl ester (38)(0.194 g, 0.471 mmol), 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimidehydrochloride (EDC) (0.108 g, 0.562 mmol) and 4-dimethylamino)pyridine(DMAP) (0.0077 g, 0.063 mmol). The mixture was stirred under argon atroom temperature for 5 hours. The solvent was removed under vacuum andthe residue was purified by silica gel column chromatography(hexane-ethyl acetate 1:1) to afford 0.24 g (0.31 mmol, 98% yield) ofproduct (20) as a syrup: ¹H NMR (CDCl₃, 200 MHz) δ 0.87 (t, 3H, J=6.3Hz), 1.25 (bs, 24H), 1.46 (s, 9H), 1.93 (m, 2H), 2.30 (m, 2H), 2.49 (m,2H), 2.80 (dd, 1H, J=16.6, 4.8 Hz), 2.93 (dd, 1H, J=16.6, 5.0 Hz),4.24–4.33 (m, 2H), 4.48–4.54 (m, 2H), 4.67–4.72 (m, 1H), 5.97 (d, 1H),7.29–7.43 (m, 5H), 7.6 (d, 2H, J=7.2 Hz), 7.76 (d, 2H, J=7.2 Hz), 9.59(bs, 1H), 10.58 (bs, 1H).

EXAMPLE 21

Preparation of the Compound of Formula (I) where X₁=S, X₂=O, R₁=—(CH₂)₁₃CH₃,

and R₃=R₄H [Compound (21)]

According to the above scheme 7 a solution of Fmoc-protected product(20) (0.120 g, 0.155 mmol) in anhydrous dichloromethane (5 ml) wastreated with 0.020 g of piperidine (0.23 mmol). The mixture was stirredat room temperature for 30 minutes. The solvent was removed under vacuumand the residue was purified by silica gel column chromatography(hexane-ethyl acetate 3:7) to afford 0.040 g (0.072 mmol, 47% yield) ofproduct (20) as a syrup: ¹H NMR (CDCl₃, 200 MHz) δ 0.87 (t, 3H, J=6.6Hz), 1.25 (bs, 24H), 1.46 (s, 9H), 1.94 (m, 2H), 2.33 (t, 2H, J=7.2 Hz),2.56 (t, 2H, J=7.7 Hz), 2.76 (d, 2H, J=5.9 Hz), 3.94 (t, 1H, J=5.8 Hz),4.21–4.30 (m, 2H).

EXAMPLE 22

Preparation of the Compound of Formula (I) where X₁=¢, X₂=O,R₁=—(CH₂)₁₃CH₃

and R₃=R₄=H [Compound (22)]

According to the above scheme 8 the tert-Butyl ester (21) (0.020 g,0.040 mmol) was treated with 0.2 ml of a 1:1 mixture of trifluoroaceticadd and dichloromethane. The mixture was stirred at room temperature for1 hour. The solvent was removed under vacuum and the residue waspurified by silica gel column chromatography (acetone-methanol, variableratios from 100:0 to 50:50) to afford 0.012 g (0.021 mmol, 54% yield) ofproduct (20) as a syrup: ¹H NMR (CD₃OD, 200 MHz) δ 0.89 (t, 3H, J=6.8Hz), 1.29 (bs, 24H), 1.97 (m, 2H), 2.35 (m, 2H), 2.57 (m, 2H), 2.82 (m,2H), 4.16–4.44 (m, 3H).

EXAMPLE 23

Preparation of the Compound of Formula (I) where X₁=S, X₂=O,R₁=—(CH₂)₁₃CH₃,

and R₃=R₄=H [Compound (23)]

wherein Bn is benzyl.

Glucose derivative (23) was prepared following a general procedure fordirect glycosilation of alcohols with glucal donor (39) (as reported in:V. Di Bussolo, Y.-J. Kim, D. Y. Gin J. Am. Chem. Soc. 1998, 120,13515–13516), as reported above in scheme 9.

Trifluoromethanesulfonic anhydride (Tf₂O) (0.030 ml, 0.18 mmol) wasadded to a solution of tri-O-benzil-D-glucal (39) (0.050 g, 0.12 mmol),diphenylsulfoxide (0.073 g, 0.36 mmol) and 2,4,6-tri-t-butylpyridine(0.104 g, 0.42 mmol) in dry chloroform (5 ml) (distilled over P₂O₅) at−40° C. The reaction mixture was stirred at this temperature for 1 hour.Methanol (0.005 ml, 0.12 mmol) and triethylamine (0.050 ml, 0.36 mmol)were added sequentially at −40° C. and the reaction mixture was stirredat this temperature for 30 minutes, then at 0° C. for 1 hour and at roomtemperature for 1 hour. A solution of alcohol derivative (17) (0.065 g,0.17 mmol) in dry chloroform (4 ml) was added at 0° C., via cannula.Zinc chloride (0.24 ml, 1.0 M in diethyl ether, 0.24 mmol) was added atthe same temperature, then the temperature was slowly warmed to roomtemperature and the reaction mixture stirred at this temperature for 12hours. The reaction was diluted with chloroform (15 ml) and washedsequentially with saturated aqueous sodium bicarbonate solution (2×15ml) and a saturated aqueous solution of sodium chloride (15 ml). Theorganic layer was dried (Na₂SO₄) and concentrated, the residue waspurified by silica gel column chromatography (hexane-ethyl acetate 6:4)to afford product (23) (0.055 g, 0.067 mmol, 56% yield) as a colourlessoil: ¹H NMR (CDCl₃) δ 0.87 (t, 3H, J=6.3 Hz), 1.25 (bs, 24H), 1.88(quintet, 2H, J=6.4 Hz), 2.44 (pseudo t, 2H, J=7.5 Hz), 2.65 (t, 2H,J=6.6 Hz), 3.70–3.66 (m, 8H), 4.47 (d, 1H, J=10.6 Hz), 4.52 (d, 1H,J=12.1 Hz), 4.65 (d, 1H, J=12.1 Hz), 4.80 (d, 1H, J=10.8 Hz), 4.86 (d,1H, J=11.4 Hz), 4.92 (d, 1H, J=11.2 Hz), 5.12 (d, 1H, J=92 Hz),7.09–7.35 (m, 15H), 9.61 (bs, 1H), 11.29 (bs, 1H); MS (FAB⁺) m/z 815(M+H)⁺.

EXAMPLE 24

Preparation of the Compound of Formula (1) Where X₁=S, X₂=O,R₁=—(CH₂)₁₃CH₃, R₂=ethyl, R₃=—CH₂COOC₂H₅, and R₄=H [Compound (24)]

Anhydrous (NH₄₂SO₄ (0.0013 g, 0.011 mmol) and1,1,1,3,3,3-hexamethyldisilazane (HMDS) (0.75 ml, 3.41 mmol) were added,under argon atmosphere, to compound (3) (0.05 g, 0.14 mmol). Theresulting suspension was heated at 130° C. and stirred at thistemperature for 6 hours. The mixture was then concentrated at roomtemperature under a flux of argon. Anhydrous THF (3 ml) was added, andthe resulting solution was stirred at −45° C. Trimethylsilyl triflate(TMS triflate) (0.03 ml, 0.145 mmol) and ethyl bromoacetate (0.046 g,0.027 mmol) were sequentially added and the mixture was stirred at −45°C. for 3 hours, then at room temperature for 1 hour. Saturated aqueousNaHCO₃ (3 ml) was added and THF was removed under vacuum. The residuewas diluted with H₂O (20 ml) and extracted with ethyl acetate (3×10 ml).The organic layer was dried with Na₂SO₄ anhydrous, and concentrated todryness. The residue was purified by semi-preparative thin-layer columnchromatography (hexane/ethyl acetate 7:3) to afford product (24) (0.010g, 0.023 mmol, 16% yield) as a colourless oil: ¹H NMR (CDCl₃, 200 MHz) δ0.87 (t, 3H, J=6.6 Hz), 1.17 (t, 3H, J=7.2 Hz), 1.25–1.43 (m, 27H), 2.44(pseudo t, 2H, J=7.2 Hz), 2.54 (t, 2H, J=7.5 Hz), 3.91 (s, 2H); 4.21 (q,2H, J=7.3 Hz), 10.88 (bs, 1H);); MS (FAB⁺) m/z 439 (M+H)⁺.

According to procedures analogues to those above reported, the followingcompounds of formula (I) were prepared:

-   -   compound (I) wherein X₁=S, X₂=O, R₁=—(CH₂)₁₃CH₃,

and R₃=R₄=H [compound (25)];

-   -   compound (I) wherein X₁=S, X₂=O, R₁=—(CH₂)₁₃CH₃, R₂=—(CH₂)₃R₄=H        [compound (26)];    -   compound (I) wherein X₁=S, X₂=O, R₁=—(CH₂)₁₃CH₃,

and R₃=R₄H [compound (27)];

-   -   compound (I) wherein X₁=S, X₂=O, R₁=—(CH₂)₁₃CH₃, R₂=—(CH₂)₃ ⁺and        R₃=R₄=H [compound (28)].        Cytotoxicity Test

The cytotoxicity of the compounds synthesized 1–28 was assessed using ahuman leukemia cell line called CCRF/CEM. The CCRF/CEM cells werecultured in a culture medium containing RPMI 1640 (90%), bovine fetalsera (10%) and interleukin-2 (100 U/ml). The cytotoxicity assay wasperformed on 104 CCRF/CEM cells seeded in 35 mm wells in 2 ml of culturemedium. The cells were treated with the compounds under considerationfor 72 hours and at the end of the period of exposure their number wascounted and compared with that of control cells treated with C₂-ceramidein order to establish the percentage of growth inhibition. Theconcentration capable of inhibiting 50% of cell growth was calculated bynon-linear regression of the experimental data as described in M.Macchia, N. Jannitti, G. B. Gervasi, R. Danesi, J Med Chem, (1996) 39(7): 1352–1356.

The resulting values of IC₅₀ expressed in μM are given in the followingtable:

Compound IC₅₀ (μM) controls 31.6  (3) 1.7  (4) 6.3  (6) 0.97  (9) 13.2(10) 8.7 (11) 20 (12) 29.1 (13) 20.7 (14) 15.6

1. Compounds of general formula (I)

where X₁ and X₂ are selected between O and S; R₁ and R₂ are selectedbetween —(CH₂)₁₃CH₃ and alkyl or alkylene groups with from 2 to 6 carbonatoms, linear or branching, unsubstituted or substituted with one ormore substituents selected among aromatic, primary, secondary andtertiary aminic, quaternary ammonium, carboxylic, hydroxylic,polyoxyalkyl and ethereal groups, amino acids, halogen atoms orsaccharides, providing that between R₁ and R₂ only one is always—(CH₂)₁₃CH₃, R₃ and R₄ are selected between H and alkyl or alkylenegroups with from 2 to 6 carbon atoms, linear or branching, unsubstitutedor substituted with one or more substituents selected among aromatic,primary, secondary and tertiary aminic, quaternary ammonium, carboxylic,hydroxylic, polyoxyalkyl and ethereal groups, amino acids, halogen atomsor saccharides.
 2. A pharmaceutical composition suitable foradministration to a mammal comprising the compound of claim 1 or apharmaceutically acceptable derivative or salt thereof, admixed with apharmaceutically acceptable excipient or diluent.
 3. A method of makinga pharmaceutical composition for the treatment of a tumor, said methodcomprising admixing the compound of claim 1 with a pharmaceuticallyacceptable excipient or diluent.
 4. A method for treating a tumor in amammal by administering the composition of claim 2.